This invention relates to a process for production of N-substituted-1-deoxynojirimycin and intermediates for its production.
A process for the preparation of 1-deoxynojirimycin in which 1-aminosorbitol is oxidized microbiologically to give 6-aminosorbose, which is then hydrogenated with a catalyst to give 1-deoxynojirimycin is disclosed in U.S. Pat. No. 4,246,345. However, the yields of this process, in particular the low volume yields in the microbiological reaction are related to degradation problems and short reaction times, in addition no process for production of N-substituted derivatives of 1-deoxynojirimycin is disclosed.
It is known that N-substituted derivatives of 1-deoxynojirimycin can be made by protecting aminosorbitols with protecting groups which are stable in subsequent microbial oxidations. The protecting groups can subsequently be removed by catalytic hydrogenation. Such a process is disclosed in U.S. Pat. No. 4,266,025. In the ""025 patent, protected amino sugars are oxidized microbiologically to give protected 6-aminosorboses, which are then isolated. The protective group is then removed by catalytic hydrogenation and the ring is reclosed to form the N-substituted derivatives of 1-deoxynojirimycin. However, the ""025 process requires a large amount of catalyst in the hydrogenation step. In addition, the unprotected 6-aminosorboses cannot be isolated as such.
U.S. Pat. No. 4,405,714 discloses a process for producing N-substituted derivatives of 1-deoxynojirimycin in which glucose is converted into a 1-aminosorbitol, the 1-aminosorbitol is then protected by a protecting group which is stable in the subsequent microbiological oxidation process. The protecting group can then be removed under acid conditions. The compounds are oxidized microbially to give a protected 6-aminosorbose. The protective group on the 6-aminosorbose is then removed under acid conditions. The 6-aminosorbose salt thus obtained is hydrogenated with a catalyst to give the N-substituted derivative of 1-deoxynojirimycin. The ""714 process, like the ""025 process, requires the use of protective groups to obtain N-substituted derivatives of 1-deoxynojirimycin.
It has been discovered that N-substituted derivatives of 1-deoxynojirimycin can be made by a process which does not require the use of protecting groups.
This invention is a process which comprises oxidizing a glucamine of the formula: 
or salts thereof, with an oxidizing microbe or extract thereof, and producing compounds of the formula: 
or salts thereof, in which R, in each instance, is phenyl, C1-C10 alkyl, C1-C10 alkyl substituted with phenyl or carboxy, or C2-C10 alkyl substituted with hydroxy.
In one embodiment of the invention, a 6-(substituted amino)-6-deoxy-xcex1-L-sorbofuranose produced by the above described reaction is reduced (with or without isolation of the intermediate sorbose) to N-substituted-1-deoxynojirimycin, or salts thereof, of formula III (shown below). 
wherein R is the same as described above.
In another embodiment of the invention, D-glucose is converted to N-substituted-1-deoxynojirimycin in a one pot process: 
Another embodiment of the invention is a process for producing N-substituted glucamines and salts thereof.
In another embodiment, the compound 6-butylamino-6-deoxy-xcex1-L-sorbofuranose and salts thereof are disclosed.
The exact form of the structure of formula II is dictated by the environment in which the sorbose is present (See H. Paulsen et al., Chem. Ber. 100:802 (1967)). The use of the sorbofuranose or the sorbose nomenclature is not meant to imply that the compound cannot or does not exist in another of its equivalent forms.
The 6-(substituted amino)-6-deoxy-xcex1-L-sorbofuranose produced by the microbial oxidation of N-substituted glucamines are useful as intermediates for producing N-substituted-1-deoxynojirimycin compounds (which are antiviral agents, antidiuretics, antidiabetics, animal feed additives and antihyperglycemics).
As used herein, the glucamines of the above formula I are referred to as xe2x80x9cN-substituted glucaminesxe2x80x9d (also known as 1-(substituted amino)-1-deoxy-D-glucitol). The compounds of the above formula II are hereinafter referred to as xe2x80x9c6-substituted amino-6-deoxy-xcex1-L-sorbofuranosesxe2x80x9d. The compounds of the above formula III are referred to as xe2x80x9cN-substituted-1-deoxynojirimycinsxe2x80x9d (also known as 1,5-(substituted imino)-1,5-dideoxy-D-glucitol and 1-substituted-3,4,5-trihydroxy-2-piperidinylmethanol.)
Straight chain or branched chain alkyls are suitable to practice the process of the invention, with C1-C5 alkyl groups preferred. Examples of suitable alkyl radicals are methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl, 1,1-dimethylethyl, n-pentyl, 3-methylbutyl, 1-methylbutyl, 2-methylbutyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl. Suitable hydroxy substituted alkyl radicals are 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl, 5-hydroxypentyl, 6-hydroxyhexyl, 7-hydroxyheptyl, 8-hydroxyoctyl, 9-hydroxynonyl, and 10-hydroxydecyl. Suitable carboxy substituted alkyl radicals are carboxymethyl, 2-carboxyethyl, 3-carboxypropyl, 4-carboxybutyl, 5-carboxypentyl, 6-carboxyhexyl, 7-carboxyheptyl, 8-carboxyoctyl, 9-carboxynonyl and 10-carboxydecyl. Suitable phenyl substituted alkyl radicals are phenylmethyl (benzyl), 2-phenylethyl, 3-phenylpropyl, 4-phenylbutyl, 5-phenylpentyl, 6-phenylhexyl, 7-phenylheptyl, 8-phenyloctyl, 9-phenylnonyl and 10-phenyldecyl. Phenyl alone is also an acceptable radical.
Examples of 6-substituted amino-6-deoxy-xcex1-L-sorbo-furanoses which are produced by the process of the invention are:
6-methylamino-6-deoxy-xcex1-L-sorbofuranose,
6-ethylamino-6-deoxy-xcex1-L-sorbofuranose,
6-n-propylamino-6-deoxy-xcex1-L-sorbofuranose,
6-(1-methylethyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-n-butylamino-6-deoxy-xcex1-L-sorbofuranose,
6-(1-methylpropyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(1,1-dimethylethyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-n-pentylamino-6-deoxy-xcex1-L-sorbofuranose,
6-(3-methylbutyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(1-methylbutyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(2-methylbutyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-n-hexylamino-6-deoxy-xcex1-L-sorbofuranose,
6-n-heptylamino-6-deoxy-xcex1-L-sorbofuranose,
6-n-octylamino-6-deoxy-xcex1-L-sorbofuranose,
6-n-nonylamino-6-deoxy-xcex1-L-sorbofuranose,
6-n-decylamino-6-deoxy-xcex1-L-sorbofuranose,
6-(2-hydroxyethyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(3-hydroxypropyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(4-hydroxybutyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(5-hydroxypentyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(6-hydroxyhexyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(7-hydroxyheptyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(8-hydroxyoctyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(9-hydroxynonyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(10-hydroxydecyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(carboxymethyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(2-carboxyethyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(3-carboxypropyl)amino-6-deoxy-xcex1-L-sorbofuranose,.
6-(4-carboxybutyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(5-carboxypentyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(6-carboxyhexyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(7-carboxyheptyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(8-carboxyoctyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(9-carboxynonyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(10-carboxydecyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-phenylamino-6-deoxy-xcex1-L-sorbofuranose,
6-(phenylmethyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(2-phenylethyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(3-phenylpropyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(4-phenylbutyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(5-phenylpentyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(6-phenylhexyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(7-phenylheptyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(8-phenyloctyl)amino-6-deoxy-xcex1-L-sorbofuranose,
6-(9-phenylnonyl)amino-6-deoxy-xcex1-L-sorbofuranose, and
6-(10-phenyldecyl)amino-6-deoxy-xcex1-L-sorbofuranose.
N-substituted glucamines of formula I can be obtained by known means, for example, by amination of D-glucose. The reductive alkylation of sugars with amines is reported in the literature as a method for preparing N-substituted-1-amino-1-deoxy sugars (see F. Kagan et al., J. Amer. Chem. Soc., 79,3541 (1957), A. Mohammad et al., J. Am. Chem. Soc., 66,969 (1947), P. N. Rylander, Hydrogenation Methods (Academic Press, (1985) pp. 82-93) and G. Mitts et al., J. Am. Chem. Soc., 66:483 (1944)). In general these preparations involve reacting a sugar and an amine, in varying ratios, in a suitable solvent such as aqueous methanol or ethanol. A catalytic amount of hydrochloric acid is sometimes added. The resulting mixture is hydrogenated under 40-1300 psig of hydrogen pressure at 23-100xc2x0 C. for 7-30 hours. The resulting 1-(substituted amino)1-deoxy D-glucitol (N-substituted glucamine) is then isolated.
In a preferred process for preparing N-substituted glucamine salts, a Parr shaker bottle, or the like, is charged with a solvent and amine. Suitable solvents include water, alcohols (such as methanol and ethanol) or aqueous alcohols. Preferably the solvent is ethanol. Suitable amines include but are not limited to the amines described for preparing the N-substituted glucamines described for formulas I, II, and III. Preferred amines include ethylamine, n-butylamine, n-octylamine, 2-hydroxyethylamine, phenylmethylamine, phenylamine and 4-carboxybutylamine. The ratio of D-glucose to amine is about 1:1, which allows the product to be used without isolation or removal of excess reagents. The mixture is stirred and cooled while acid is slowly added until a pH in the range of about 8.0 to 12.0 is obtained, preferably about 9 to 10.5. Suitable acids include hydrochloric acid, sulphuric acid, nitric acid, acetic acid, ascorbic acid, succinic acid, citric acid, maleic acid, oxalic acid, and phosphoric acid, preferably hydrochloric acid. To the Parr shaker bottle is added D-glucose followed by palladium-on-carbon (Pd/C) catalyst (50% water-wet). A palladium catalyst loading of about 1% to 50% by weight glucose is used, preferably about 10% to 30%. Catalysts from the noble metals can be used, for example, platinum, palladium, rhodium and rhenium, preferably palladium. The mixture is agitated and hydrogenated at a pressure of about 1 to 100 atm, preferably about 3-6 atm of hydrogen at a temperature of about 25xc2x0 C. to 100xc2x0 C., preferably about 40xc2x0 C. to 80xc2x0 C., until the reaction is complete (as indicated by hydrogen uptake). The hydrogen is vented and the palladium-on-carbon removed by filtration (preferably through a layer of powdered cellulose). The catalyst is washed with solvent such as an alcohol, preferably ethanol, followed by washing with water. The washes are combined with the filtrate to give a solution containing a mixture of N-substituted glucamine and its corresponding salt. The mixture is stirred and cooled while hydrochloric acid is slowly added to a final pH of about 1 to 7, preferably about 4 to 6. The ethanol is removed by distillation under reduced pressure. The residue contains the N-substituted glucamine salt. The residue is diluted with water and ready to use in the next step of microbial oxidation without purification. Thus, the process produces the N-substituted glucamine salts from D-glucose without isolation or removal of excess reagents. The elimination of isolation and excess reagent removal steps allows for the direct use of N-substituted glucamines in the microbial oxidation, which oxidation results in the 6-substituted amino-6-deoxy-xcex1-L-sorbofuranoses which in turn can be directly hydrogenated to N-substituted-1-deoxynojirimycins (i.e. one pot process).
An additional advantage of the N-substituted glucamine salts is the elimination of odor associated with residual amines. Typically the amines are extremely odoriferous, requiring the use of respirators when handling. On the other hand, the glucamine salts are relatively odor free, which enables handling without special precautions such as respirators.
As indicated by Material Safety Data Sheets from suppliers of N-n-butylamine (Fisher Scientific, Fair Lawn, N.J., for example), the N-n-butylamine compound is toxic and a severe eye, skin and mucous membrane irritant. Exposure to as little as 5-10 ppm of N-butylamine produces nose and throat irritation. Exposure to concentrations of 10-25 ppm are intolerable for more than a few minutes. Thus, the salt forms of the N-substituted glucamine compounds, which forms do not have the odor and irritation characteristics of the non-salt forms are advantageous.
To begin the microbial oxidation of an N-substituted glucamine, oxidizing microorganisms are added to a reaction mixture which comprises an N-substituted glucamine or salts thereof. Alternatively, N-substituted glucamine or a salt thereof is added to cultures of oxidizing microorganisms that will carry out the oxidation step. Preferably a salt of N-substituted glucamine is added. Suitable salts of N-substituted glucamine include but are not limited to salts of chloride, sulphate, nitrate, acetate, ascorbate, succinate, citrate, maleate, oxalate, or phosphate. Preferably the hydrochloride salt is used. Although the use of a salt is preferred, a salt can be made in situ by the addition of an N-substituted glucamine and suitable acids to lower the pH and create an N-substituted glucamine salt. During incubation of the reaction mixture containing microorganisms, the reaction is monitored with a reverse phase or ion exchange high performance liquid chromatography (HPLC) assay to observe conversion of N-substituted glucamine to the respective 6-(substituted amino)-6-deoxy-xcex1-L-sorbofuranose. Thin layer chromatography (TLC), and gas chromatography (GC) can also be used to monitor the conversion.
Oxidizing microorganisms which are suitable for carrying out the oxidation (or microorganisms from which active extracts for carrying out the oxidation are obtained) can be Procaryotae (bacteria), or Eucaryotae, for example fungi, which in each case can belong to diverse taxonomic groups. Suitable microorganisms are found by growing a relatively large number of microorganisms in an appropriate nutrient medium which contains sorbitol or N-substituted glucamines and examining their ability to produce sorbose or 6-(substituted amino)-6-deoxy-xcex1-L-sorbofuranoses, respectively. The ability of a microorganism to catalyze the oxidation reaction according to the invention can be measured by a variety of means, including assaying with high performance liquid chromatography (HPLC). Microorganisms for use in the process of the invention are readily available from a variety of sources including but not limited to the American Type Culture Collection (ATCC), Rockville, Md.; the Agricultural Research Culture Collection (NRRL), Peoria, Ill.; Deutsche Sammlung Von Mikroorganismen (DSM), Federal Republic of Germany; and the Fermentation Research Institute (FRI), Japan.
Examples of suitable oxidizing microorganisms are bacteria from the order Pseudomonadales, bacteria from the family Pseudomonadaceae, bacteria from the family Coryneform, and fungi from the genus Metschnikowia. Within the Pseudomonadales order, preference is for representatives of the family Pseudomonadaceae. Within the Pseudomonadaceae family, bacteria of the genus Gluconobacter (formerly called Acetobacter) are preferred. Bacteria from the group of Coryneform bacteria, in particular those of the genus Corynebacterium (also known as Curtobacterium), are also suitable. Finally, the oxidation can be carried out with fungi (for example, with yeasts) in particular with those of the family Spermophthoraceae, such as the genus Metschnikowia.
Examples of suitable Corynebacterium are Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium acnes, Corynebacterium alkanolyticum, Corynebacterium alkanum, Corynebacterium betae, Corynebacterium bovis, Corynebacterium callunae, Corynebacterium cystitidis, Corynebacterium dioxydans, Corynebacterium equi, Corynebacterium flavescens, Corynebacterium glutamicum, Corynebacterium herculis, Corynebacterium hoagii, Corynebacterium hydrocarbooxydans, Corynebacterium ilicis, Corynebacterium lilium, Corynebacterium liquefaciens, Corynebacterium matruchotii, Corynebacterium melassecola, Corynebacterium mycetoides, Corynebacterium nephridii, Corynebacterium nitrilophilus, Corynebacterium oortii, Corynebacterium petrophilum, Corynebacterium pilosum, Corynebacterium pyogenes, Corynebacterium rathayi, Corynebacterium renale, Corynebacterium simplex, Corynebacterium striatum, Corynebacterium tritici, Corynebacterium uratoxidans, Corynebacterium vitarumen, and Corynebacterium xerosis. Suitable Gluconobacterium for use in the process of the invention include Gluconobacter oxydans subsp. industrius, Gluconobacter oxydans subsp. melanogenes, Gluconobacter oxydans subsp. sphaericus, and Glucanobacter oxydans subsp. suboxydans. Metschnikowia (formerly called Candida) preferred for use in the process of the invention include Metschnikowia pulcherrimia. 
General growth conditions for culturing the particular organisms are obtained from depositories and from texts known in the art such as Bergey""s Manual of Systematic Bacteriology, Vol.1, Williams and Wilkins, Baltimore/London (1984), N. R. Krieg, ed.
The nutrient medium for the growth of any oxidizing microorganism should contain sources of assimilable carbon and nitrogen, as well as mineral salts. Suitable sources of assimilable carbon and nitrogen include, but are not limited to, complex mixtures, such as those constituted by biological products of diverse origin, for example soy bean flour, cotton seed flour, lentil flour, pea flour, soluble and insoluble vegetable proteins, corn steep liquor, yeast extract, peptones and meat extracts. Additional sources of nitrogen are ammonium salts and nitrates, such as ammonium chloride, ammonium sulphate, sodium nitrate and potassium nitrate. Generally, the nutrient medium should include, but is not limited to, the following ions: Mg++, Na+, K+, Ca++, NH4+. Clxe2x88x92, SO4xe2x88x92xe2x88x92, PO4xe2x88x92xe2x88x92 and NO3xe2x88x92 and also ions of the trace elements such as Cu, Fe, Mn, Mo, Zn, Co and Ni. The preferred source of these ions are mineral salts.
If these salts and trace elements are not present in sufficient amounts in the complex constituents of the nutrient medium or in the water used it is appropriate to supplement the nutrient medium accordingly.
The microorganism employed in the process of the invention can be in the form of fermentation broths, whole washed cells, concentrated cell suspensions, enzyme extracts, and immobilized cells. Preferably concentrated cell suspensions, enzyme extracts, and whole washed cells are used with the process of the invention (S. A. White and G. W. Claus (1982), J. Bacteriology 150: 934-943).
Concentrated washed cell suspensions may be prepared as follows: The microorganisms are cultured in a suitable nutrient solution, harvested (for example by centrifuging) and suspended in a smaller volume (in salt or buffer solutions, such as physiological sodium chloride solution or aqueous solutions of potassium phosphate, sodium acetate, sodium maleate, magnesium sulfate, or simply in tap water, distilled water or nutrient solutions). N-substituted glucamine or a salt thereof is then added to a cell suspension of this type and the oxidation reaction according to the invention is carried out under the conditions described.
The conditions for oxidation of N-substituted glucamine in growing microorganism cultures or fractionated cell extracts are advantageous for carrying out the process according to the invention with concentrated cell suspensions. In particular the temperature range is from about 0xc2x0 C. to about 45xc2x0 C. and the pH range is from about 2 to about 10. There are no special nutrients necessary in the process of the invention. More importantly, washed or immobilized cells can simply be added to a solution of N-substituted glucamine or salts thereof, without any nutrient medium present.
It is also possible to carry out the process according to the invention with enzyme extracts or enzyme extract fractions prepared from bacteria. The extracts can be crude extracts, such as obtained by conventional digestion of microorganism cells. Methods to break up cells include, but are not limited to, mechanical disruption, physical disruption, chemical disruption, and enzymatic disruption. Such means to break up cells include ultrasonic treatments, passages through French pressure cells, grindings with quartz sand, autolysis, heating, osmotic shock, alkali treatment, detergents, or repeated freezing and thawing.
If the process according to the invention is to be carried out with partially purified enzyme extract preparations, the methods of protein chemistry, such as ultracentrifuging, precipitation reactions, ion exchange chromatography or adsorption chromatography, gel filtration or electrophoretic methods, can be employed to obtain such preparations. In order to carry out the reaction according to the invention with fractionated cell extracts, it may be necessary to add to the assay system additional reactants such as, physiological or synthetic electron acceptors, like NAD+, NADP+, methylene blue, dichlorophenolindophenol, tetrazolium salts and the like. When these reactants are used, they can be employed either in equimolar amounts (concentrations which correspond to that of the N-substituted glucamine employed) or in catalytic amounts (concentrations which are markedly below the chosen concentration of N-substituted glucamine). If, when using catalytic amounts, it is to be ensured that the process according to the invention is carried out approximately quantitatively, a system which continuously regenerates the reactant which is present only in a catalytic amount must also be added to the reaction mixture. This system can be, for example, an enzyme which ensures reoxidation (in the presence of oxygen or other oxidizing agents) of an electron acceptor which is reduced in the course of the reaction according to the invention.
If nutrient media is used with intact microorganisms in a growing culture, nutrient media can be solid, semi-solid or liquid. Aqueous-liquid nutrient media are preferably employed when media is used. Suitable media and suitable conditions for cultivation include known media and known conditions to which N-substituted glucamine or salts thereof can be added.
The N-substituted glucamine or salts thereof to be oxidized in the process according to the invention can be added to the base nutrient medium either on its own or as a mixture with one or more oxidizable compounds. Additional oxidizable compounds which can be used include polyols, such as sorbitol or glycerol.
If one or more oxidizable compounds are added to the nutrient solution, the N-substituted glucamine or salts thereof to be oxidized can be added either prior to inoculation or at any desired subsequent time (between the early log phase and the late stationary growth phase). In such a case the oxidizing organism is pre-cultured with the oxidizable compounds. The inoculation of the nutrient media is effected by a variety of methods including slanted tube cultures and flask cultures.
Contamination of the reaction solution should be avoided. To avoid contamination, sterilization of the nutrient media, sterilization of the reaction vessels and sterilization of the air required for aeration should be undertaken. It is possible to use, for example, steam sterilization or dry sterilization for sterilization of the reaction vessels. The air and the nutrient media can likewise be sterilized by steam or by filtration. Heat sterilization of the reaction solution containing the substrates (N-substituted glucamine) is also possible.
The process of the invention can be carried out under aerobic conditions using shake flasks or aerated and agitated tanks. Preferably, the process is carried out by the aerobic submersion procedure in tanks, for example in conventional fermentors. It is possible to carry out the process continuously or with batch or fed batch modes, preferably the batch mode.
It is advantageous to ensure that the microorganisms are adequately brought into contact with oxygen and the N-substituted glucamines. This can be effected by several methods including shaking, stirring and aerating.
If foam occurs in an undesired amount during the process, chemical foam control agents, such as liquid fats and oils, oil-in-water emulsions, paraffins, higher alcohols (such as octadecanol), silicone oils, polyoxyethylene compounds and polyoxypropylene compounds, can be added. Foam can also be suppressed or eliminated with the aid of mechanical devices.
The time-dependent formation of 6-(substituted amino)-6-deoxy-xcex1-L-sorbofuranose in the culture medium can be followed either by thin layer chromatography, HPLC, or with the aid of the increase in the inhibitory activity in the saccharase inhibition test. Preferably the time-dependent formation of 6-(substituted amino)-6-deoxy-xcex1-L-sorbose is measured by HPLC.
The 6-(substituted amino)-6-deoxy-xcex1-L-sorbofuranose obtained in accordance with the process of the invention is isolated from the reaction solution as follows: The cell mass is filtered off or centrifuged off and the supernatant liquor is passed through a column containing acid ion exchanger and rinsed with an alcohol or water. Elution is then carried out with a base and the eluate concentrated. After adding acetone or the like, the 6-(substituted amino)-6-deoxy-xcex1-L-sorbose crystallizes out. If it is intended to carry out further processing of 6-(substituted amino)-6-deoxy-xcex1-L-sorbose to N-substituted-1-deoxynojirimycin, isolation is not necessary. For producing N-substituted-1-deoxynojirimycin from 6-(substituted amino)-6-deoxy-xcex1-L-sorbofuranose, the clear solution, after removal of the cell mass, is reduced, preferably in the presence of a catalyst.
This aspect of the invention (no isolation necessary) is particularly advantageous because the process proceeds directly from the supernatant liquor resulting from the removal of cell mass of the microbial oxidation reaction solution. Likewise, it is especially advantageous because, unlike prior art processes, no amino protecting group has to be removed. The process of the invention eliminates the need to make and isolate protecting group intermediates and avoids removal of the protecting group to obtain the desired compound. The elimination of these steps results in a more efficient process with greater conversions and overall yields. The 6-(substituted amino)-6-deoxy-xcex1-L-sorbofuranoses also exhibit higher solubility, thus higher concentrations are obtainable, which results in high productivity and higher rates. In addition, the 6-(substituted amino)-deoxy-xcex1-L-sorbofuranoses have great stability which impedes degradation and resulting byproducts.
Several known means are available for reduction (see for example P. N. Rylander, Hydrogenation Methods (Academic Press, (1985) pp 82-93 and Organic Chemistry, 3rd edition, Eds James B. Hendrickson, Donald J. Cram, George S. Hammond (McGraw-Hill, Chapter 18, 1970)). These means include metal hydride reduction, catalytic hydrogenation, dissolving metal reduction and electrochemical reduction. In general, to reduce 6-(substituted amino)-6-deoxy-xcex1-L-sorbofuranose to N-substituted-1- deoxynojirimycin, 6-(substituted amino)-6-deoxy-xcex1-L-sorbofuranose is charged to a flask followed by addition of decolorizing carbon. The stirred mixture is then filtered to remove the carbon. The filtrate is added to a hydrogenation apparatus, such as a Parr Laboratory Reactor, containing a hydrogenation catalyst. Catalyst loading from about 1-50% by weight of sorbofuranose using Group VIII B metals are used. Preferably about 10-30% is used. Such catalysts include but are not limited to palladium, platinum, nickel and rhodium. Supports for the catalysts may include but are not limited to alumina, barium sulfate, calcium carbonate, carbon, silica and kieselguhr. Typically, the support would contain a 1-20% metal loading, preferably a 4-10% loading. A palladium catalyst is preferred. However, the preferred catalyst for 6-phenylmethylamino-6-deoxy-xcex1-L-sorbofuranose is platinum or Raney nickel. The mixture is hydrogenated for about 5 hours. Hydrogen pressure from about 1 to 100 atm can be used; preferably a range from about 1-5 atm is used. The catalyst is then removed and acid ion-exchange resin added to the filtrate to adsorb the N-substituted-1-deoxynojirimycin. The N-substituted-1-deoxynojirimycin is released from the resin and isolated.
Examples of N-substituted-1-deoxynojirimycins that can be produced by hydrogenating the 6-(substituted amino)-6-deoxy-xcex1-L-sorbofuranose produced by the microbial oxidation process of the invention are:
N-methyl-1-deoxynojirimycin,
N-ethyl-1-deoxynojirimycin,
N-n-propyl-1-deoxynojirimycin,
N-1-methylethyl-1-deoxynojirimycn,
N-n-butyl-1-deoxynojirimycin,
N-1-methylpropyl-1-deoxynojirimycin,
N-1,1-dimethylethyl-1-deoxynojirimycin,
N-n-pentyl-1-deoxynojirimycin,
N-3-methylbutyl-1-deoxynojirimycin,
N-1-methylbutyl-1-deoxynojirimycin,
N-2-methylbutyl-1-deoxynojirimycin,
N-n-hexyl-1-deoxynojirimycin,
N-n-heptyl-1-deoxynojirimycin,
N-n-octyl-1-deoxynojirimycin,
N-n-nonyl-1-deoxynojirimycin,
N-n-decyl-1-deoxynojirimycin,
N-(2-hydroxyethyl)-1-deoxynojirimycin,
N-(3-hydroxypropyl)-1-deoxynojirimycin,
N-(4-hydroxybutyl)-1-deoxynojirimycin,
N-(5-hydroxypentyl)-1-deoxynojirimycin,
N-(6-hydroxyhexyl)-1-deoxynojirimycin,
N-(7-hydroxheptyl)-1-deoxynojirimycin,
N-(8-hydroxyoctyl)-1-deoxynojirimycin,
N-(9-hydroxynonyl)-1-deoxynojirimycin,
N-(10-hydroxydecyl)-1-deoxynojirimycin,
N-(carboxymethyl)-1-deoxynojirimycin,
N-(2-carboxyethyl)-1-deoxynojirimycin,
N-(3-carboxypropyl)-1-deoxynojirimycin,
N-(4-carboxybutyl)-1-deoxynojirimycin,
N-(5-carboxypentyl)-1-deoxynojirimycin,
N-(6-carboxyhexyl)-1-deoxynojirimycin,
N-(7-carboxyheptyl)-1-deoxynojirimycin,
N-(8-carboxyoctyl)-1-deoxynojirimycin,
N-(9-carboxynonyl)-1-deoxynojirimycin,
N-(10-carboxydecyl)-1-deoxynojirimycin,
N-phenyl-1-deoxynojirimycin,
N-(phenylmethyl)-1-deoxynojirimycin,
N-(2-phenylethyl)-1-deoxynojirimycin,
N-(3-phenylpropyl)-1-deoxynojirimycin,
N-(4-phenylbutyl)-1-deoxynojirimycin,
N-(5-phenylpentyl)-1-deoxynojirimycin,
N-(6-phenylhexyl)-1-deoxynojirimycin,
N-(7-phenylheptyl)-1-deoxynojirimycin,
N-(8-phenyloctyl)-1-deoxynojirimycin,
N-(9-phenylnonyl)-1-deoxynojirimycin, and
N-(10-phenyldecyl)-1-deoxynojirimycin.
The following examples illustrate the specific embodiments of the invention described herein. As would be apparent to skilled artisans, various changes and modifications are possible and are contemplated within the scope of the invention described.
A Gluconobacter oxydans cell paste is prepared by inoculating a series of 10 liter fermentors, each containing eight liters of media with 60 gm./liter D-sorbitol, 24 gm./liter yeast extract, 48 gm./liter potassium phosphate dibasic and 0.3 ml./liter antifoam (Ucon LB 625) with the microorganism G. oxydans (DSM2003). The fermentors are agitated and aerated while controlling temperature (30xc2x0 C.) and pH (5.5 to 6.5) during the cell growth period. The fermentations are terminated after about 27 hours when optical density measurements indicate the log growth phase has been completed. The broths are then cooled, centrifuged, and the cells resuspended in water (or 0.02M MgSO4) and centrifuged to produce washed cell paste. These cell pastes are subdivided into aliquots and stored at or below 10xc2x0 C. until thawed for addition to a reaction solution.
The reaction solution can also be sterilized by autoclaving, as described below. A 5% (weight/volume) solution of N-butylglucamine containing 20 mM MgSO4, at pH 5.0, was autoclaved in a 500 ml shake flask with a silicon closure in place. The conditions for autoclaving were 121xc2x0 C., 30 P.S.I., for 30 minutes. The pH was checked afterwards and found to be 5.0 at room temperature. There was no visible change in the color or the clarity of the solution, an indication that no caramelization or precipitation had occurred. Analysis by HPLC assay also showed that the reaction solutions before and after autoclaving were the same. Gluconobacter oxydans cells were added to the shake flask and HPLC results showed conversion of at least 90% after 48 hours. Controls (not autoclaved) indicated at least 90% conversion at 48 hours, also. Autoclaving can therefore be used as a method for sterilizing the reaction solution.